Recombinant immune cells, methods of making, and methods of use

ABSTRACT

A recombinant immune cell expresses a heterologous IgG Fc receptor. In some embodiments, the heterologous IgG Fc receptor can be a chimeric IgG Fc receptor. Generally, the chimeric IgG Fc receptor includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain generally includes a sufficient portion of CD64 to bind to an IgG Fc region. The intracellular domain of the chimeric IgG Fc receptor includes a sufficient portion of an Fc receptor allowing immunoreceptor tyrosine-based activation motif (ITAM) to initiate cell signaling when an IgG Fc region binds to the extracellular domain.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/577,425, filed Oct. 26, 2017, which is incorporated herein byreference in its entirety.

GOVERNMENT FUNDING

This invention was made with government support under CA203348 awardedby National Institutes of Health. The government has certain rights inthe invention.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submitted tothe United States Patent and Trademark Office via EFS-Web as an ASCIItext file entitled “Sequence-Listing-0589_ST25.txt” having a size of 97kilobytes and created on Oct. 26, 2018. Due to the electronic filing ofthe Sequence Listing, the electronically submitted Sequence Listingserves as both the paper copy required by 37 CFR § 1.821(c) and the CRFrequired by § 1.821(e). The information contained in the SequenceListing is incorporated by reference herein.

SUMMARY

This disclosure describes, in one aspect, this disclosure describes animmune cell that expresses a heterologous IgG Fc receptor.

In some embodiments, the heterologous IgG Fc receptor can be a chimericIgG Fc receptor. Generally, the chimeric IgG Fc receptor includes anextracellular domain, a transmembrane domain, and an intracellulardomain. The extracellular domain generally includes a sufficient portionof CD64 to bind to an IgG Fc region. The intracellular domain of thechimeric IgG Fc receptor includes a sufficient portion of an Fc receptorimmunoreceptor tyrosine-based activation motif (ITAM) to initiate cellsignaling when an IgG Fc region binds to the extracellular domain.

In some of these embodiments, the intracellular domain includes at leasta portion of the intracellular region of CD16A. In other embodiments,the intracellular domain can include at least a portion of theintracellular region of CD27, CD28, CD134 (OX40), CD137 (4-1BB), FcRγ,or CD3.

In some embodiments, the chimeric IgG Fc receptor can include the CD16Aextracellular cleavage site. In other embodiments, the extracellulardomain of the chimeric IgG Fc receptor can lack the CD16A extracellularcleavage site.

In some embodiments, the heterologous IgG Fc receptor can include an IgGFc receptor not natively expressed by the immune cell. In some of theseembodiments, the immune cell may be a natural killer (NK) cellgenetically modified to express CD64.

In another aspect, this disclosure describes a polynucleotide thatencodes any embodiment of the heterologous IgG Fc receptors summarizedabove.

In another aspect, this disclosure describes an immune cell that isgenetically modified to include the polynucleotide that encodes anembodiment of the heterologous IgG Fc receptors summarized above.

In another aspect, this disclosure describes a method of killing a tumorcell. Generally, the method includes contacting the tumor cell with anantibody that specifically binds to the tumor cell and contacting thetumor cell with any embodiment of the recombinant immune cell summarizedabove under conditions effective for the recombinant immune cell to killthe tumor cell.

In another aspect, this disclosure describes a method of treating asubject having a tumor. Generally, the method includes administering tothe subject an antibody that specifically binds to cells of the tumorand administering to the subject a composition that includes anyembodiment of the recombinant immune cell summarized above underconditions effective for the recombinant immune cell to kill cells ofthe tumor.

In another aspect, this disclosure describes a composition that includea complex formed between a therapeutic antibody and any embodiment ofthe recombinant immune cell summarized above in which the heterologousIgG Fc receptor is bound to the Fc portion of the therapeutic antibody.

In another aspect, this disclosure describes a method of treating asubject having a tumor. Generally, the method includes administering tothe subject any embodiment of the composition summarized immediatelywherein the therapeutic antibody specifically binds to cells of thetumor.

The above summary is not intended to describe each disclosed embodimentor every implementation of the present invention. The description thatfollows more particularly exemplifies illustrative embodiments. Inseveral places throughout the application, guidance is provided throughlists of examples, which examples can be used in various combinations.In each instance, the recited list serves only as a representative groupand should not be interpreted as an exclusive list.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. Antibody-dependent cell-mediated cytotoxicity (ADCC).

FIG. 2. Wildtype CD16A, wildtype CD64, and the CD64/16A chimericconstruct. The scissors and dashed line shown for CD16A represent theextracellular proteolytic site for ectodomain shedding.

FIG. 3. NK92 cells expressing either wildtype CD16A or CD64/16A. (A)NK92-CD64/16A cells were stained with anti-CD16, anti-CD64, or controlantibodies. (B) NK92-CD16A cells were stained with anti-CD16, anti-CD64,or control antibodies. (C) NK92-CD64/16A, NK92-CD16A, or NK92 parentcells were incubated with trastuzumab then anti-human IgG-APC secondstage antibody or anti-human IgG-APC second stage antibody alone(control). All antibody staining levels were determined by flowcytometry.

FIG. 4. NK92 cells expressing CD64/CD16A induce higher levels of ADCCthan wildtype CD16A. (A) A standard ADCC assay was performed in whichtrastuzumab (herceptin) was included in the assay. (B) A standard ADCCassay was performed in which the NK92-CD64/CD16A and NK92-CD16A cellswere pre-incubated with trastuzumab and then the mAb was washed awayprior to the effector cells being incubated with the SKOV-3 targetcells.

FIG. 5. Flow cytometry data comparing phenotypic markers expressed byiNK-CD64/CD16A and iNK-pKT2 cells.

FIG. 6. Bar graph with data showing iNK-CD64/CD16A cells induce higherlevels of ADCC than iNK-CD16A cells using SKOV-3 target cells.

FIG. 7. Bar graph with data showing iNK-CD64/CD16A, but not iNK-CD16Acells can be pre-loaded with a therapeutic mAb and mediate ADCC.

FIG. 8. Bar graph with data showing iNK-CD64/CD16A cells induce higherlevels of ADCC than iNK-CD16A cells using MA148 target cells.

FIG. 9. Amino acid sequence of an exemplary CD64/CD16A chimeric IgG Fcreceptor (SEQ ID NO:1).

FIG. 10. Amino acid sequence of CD64 IgG Fc receptor (SEQ ID NO:2).

FIG. 11. Amino acid sequence of an exemplary CD16A-CD28-BB-ζ chainchimeric IgG Fc receptor (SEQ ID NO:3).

FIG. 12. Amino acid sequence of an exemplary CD16A-BB-ζ chain chimericIgG Fc receptor (SEQ ID NO:4).

FIG. 13. Expression of CD64/16A by NK92 cells. (A) Schematicrepresentation of the cell membrane forms of CD16A, CD64, and CD64/16A.CD16A undergoes ectodomain shedding by ADAM17 at a membrane proximallocation, as indicated, which is not present in CD64 and CD64/16A. (B)NK92 parental cells, NK92-CD16A cells, and NK92-CD64/16A cells werestained with an anti-CD16, anti-CD64, or an isotype-matched negativecontrol mAb and examined by flow cytometry. (C) NK92-CD16A andNK92-CD64/16A cells were incubated with SKOV-3 cells with or withouttrastuzumab (5 μg/ml) at 37° C. (E:T=1:1) for two hours. The NK92-CD16Aand NK92-CD64/16A cells were then stained with an anti-CD16 mAb or ananti-CD64 mAb, respectively, and examined by flow cytometry. Nonspecificantibody labeling was determined using the appropriate isotype-negativecontrol mAb. Data is representative of at least three independentexperiments.

FIG. 14. CD64/16A promotes target cell conjugation, ADCC, and IFNγproduction. (A) NK92-CD64/16A cells expressing eGFP and SKOV-3 cellslabeled CellTrace Violet were mixed at an E:T ratio of 1:2 with orwithout trastuzumab (5 μg/ml), incubated at 37° C. for 60 minutes,fixed, and then analyzed by flow cytometry. Representative data from atleast three independent experiments are shown. (B) NK92-CD64/16A cellswere incubated with SKOV-3 cells (E:T=20:1) and trastuzumab (tras.) atthe indicated concentrations (left panel), or with SKOV-3 cells at theindicated E:T ratios in the presence or absence of trastuzumab (5 μg/ml)(right panel) for two hours at 37° C. Data are represented as % specificrelease and the mean±SD of three independent experiments is shown.Statistical significance is indicated as *p<0.05, **p<0.01. (C)NK92-CD64/16A cells were incubated with SKOV-3 cells (E:T=20:1) in thepresence or absence of trastuzumab (5 μg/ml) and the anti-CD64 mAb 10.1(10 μg/ml), as indicated, for two hours at 37° C. Data are representedas % specific release and the mean±SD of three independent experimentsis shown. Statistical significance is indicated as **p<0.01. (D)NK92-CD64/16A cells were incubated with SKOV-3 cells (E:T=1:1) with orwithout trastuzumab (5 μg/ml) for two hours at 37° C. Secreted IFNγlevels were quantified by ELISA. Data is shown as mean of twoindependent experiments.

FIG. 15. CD64/16A attaches to soluble tumor-targeting mAbs and IgGfusion proteins. (A) Relative expression levels of CD16A and CD64/16A onNK92 cells were determined by cell staining with anti-CD16 and anti-CD64mAbs (black bars), respectively, or an isotype-matched negative controlantibody (gray bars). The bar graph shows mean fluorescence intensity(MFI)±SD of three independent experiments. Representative flowcytometric data are shown in the histogram overlay. The dashed linehistogram shows CD64 staining of NK92-CD64/16A cells, the orange-filledhistogram shows CD16A staining of NK92-CD16A cells, and the green-filledhistogram shows isotype control antibody staining of the NK92-CD16Acells. (B) NK92-CD16A and NK92-CD64/16A cells were incubated with orwithout trastuzumab (5 μg/ml) for two hours at 37° C., washed, stainedwith a fluorophore-conjugated anti-human secondary antibody, andanalyzed by flow cytometry. Data is representative of at least threeindependent experiments. (C) NK92-CD64/16A cells were incubated withcetuximab or rituximab (5 μg/ml for each), washed, and then stained witha fluorophore-conjugated anti-human secondary antibody. Controlrepresents cells stained with the anti-human secondary antibody only.NK92-CD64/16A cells were also incubated with L-selectin/Fc (5 μg/ml),washed, and then stained with a fluorophore-conjugated anti-L-selectinmAb. NK92 cells lack expression of endogenous L-selectin (data notshown). All staining was analyzed by flow cytometry. Data shown arerepresentative of three independent experiments. (D) NK92-CD16A andNK92-CD64/16A cells were incubated in the presence or absence oftrastuzumab (5 μg/ml), washed, and exposed to SKOV-3 cells at theindicated E:T cell ratios for two hours at 37° C. Data is shown asmean±SD of three independent experiments. Statistical significance isindicated as **p<0.01, ***p<0.001. bd=below detection, (i.e.,<spontaneous release by negative control cells). (E) NK92-CD16A andNK92-CD64/16A cells were incubated with SKOV-3 cells (E:T=10:1) in thepresence or absence of trastuzumab (5 μg/ml), as indicated, for twohours at 37° C. Data is shown as mean±SD of three independentexperiments. Statistical significance is indicated as **p<0.01.

FIG. 16. Generation of iNK cells expressing CD64/CD16A. iPSCs weretransduced to stably express CD64/16A, differentiated into NK cells, andthen expanded using K562-mbIL21-41BBL feeder cells. iNK-CD64/16A cellsand freshly isolated peripheral blood (PB) NK cells enriched from adultperipheral blood were stained for CD56, CD3 and various inhibitory andactivating receptors, as indicated. CD64/16A expression was determinedby staining the cells with an anti-CD64 mAb. Representative data from atleast three independent experiments are shown.

FIG. 17. iNK-CD64/16A cells show enhanced ADCC compared to iNK-pKT2control cells. (A) NK cells derived from empty vector (iNK-pKT2) orCD64/16A (iNK-CD64/16A) transduced iPSCs were stained for CD56, CD64,and CD16A, as indicated. (B) iNK-pKT2 and iNK-CD64/16A cells wereincubated with SKOV-3 cells (E:T=10:1) in the presence or absence oftrastuzumab (5 μg/ml), the function blocking anti-CD16 mAb 3G8 (5μg/ml), and the function blocking anti-CD64 mAb 10.1 (5 μg/ml), asindicated, for two hours at 37° C. Data is shown as mean±SD of threeindependent experiments. Statistical significance is indicated as***p<0.001; ****p<0.0001. (C) iNK-pKT2 and iNK-CD64/16A cells wereincubated in the presence or absence of trastuzumab (5 μg/ml), washed,and exposed to SKOV-3 cells (E:T=10:1) for two hours at 37° C. Data isshown as mean±SD of three independent experiments. Statisticalsignificance is indicated as ***p<0.001.

FIG. 18. Sequence alignment of canine CD16A (SEQ ID NO:5), canine CD64sp(SEQ ID NO:25), and human CD16A (SEQ ID NO:6).

FIG. 19. Sequence alignment of canine CD64 (SEQ ID NO:7) and human CD64(SEQ ID NO:8).

FIG. 20. NK92 cells expressing wildtype human CD64 mediate ADCC. (A)NK92-CD64 cells were stained with an isotype-matched negative controlmAb or the anti-CD64 mAb (clone 10.1) and examined by flow cytometry.(B) NK92-CD64 cells were incubated with SKOV-3 cells (at the indicatedE:T ratios) in the presence or absence of trastuzumab (tras.) (5 μg/ml)for two hours at 37° C. Representative data from at least threeindependent experiments are shown.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

This disclosure describes recombinant immune cells, methods of makingthe recombinant immune cells, and methods of using the recombinantimmune cells. Generally, the recombinant immune cells are geneticallymodified to include a heterologous IgG Fc receptor. In some cases, theheterologous IgG Fc receptor can be a chimeric receptor engineeredinclude domains from two or more receptors. In other embodiments, theheterologous may be an IgG Fc receptor that is not natively expressed bythe immune cell. Generally, the recombinant immune cells provide asustained cytotoxic immune response against a target—for example, atumor cell—that is targeted for killing by the immune cell because thetarget binds a therapeutic antibody that is recognized by the IgG Fcreceptor.

A mechanism of cell-mediated immune defense involves the engagement ofantibodies attached to target cells by Fc receptors expressed byleukocytes, which results in target cell killing. This process isreferred to antibody-dependent cell-mediated cytotoxicity (ADCC).Therapeutic monoclonal antibodies (mAbs) have been generated against avariety of tumor antigens and tested in clinical trials for treatinginfectious diseases, chronic diseases, and cancers including, forexample, AML, breast cancer, ovarian cancer, gastric cancer,neuroblastoma, and lymphoma. Many clinically successful mAbs use ADCC asa mechanism of action. A limitation of antibody therapy, however, is thedevelopment of resistance in patients and the non-responsiveness of somemalignancies.

This disclosure describes an approach for augmenting Fc receptorinteractions with therapeutic antibodies. The approach involves achimeric receptor that includes a CD16A domain and a CD64 domain.

CD16A (FcγRIIIA) is an IgG Fc receptor expressed by human natural killer(NK) cells, a population of cytotoxic lymphocytes, and is their solemeans of recognizing IgG bound to tumor cells or virus-infected cells.CD16A is a potent activating receptor that induces ADCC by NK cells(FIG. 1). The CD16A transmembrane region is responsible for theassociation with CD3 and/or FcR γ-chain (FcRγ) that containimmunoreceptor tyrosine-based activation motifs (FIG. 2; FIG. 13A), andthe CD16A cytoplasmic domain interacts with intracellular moleculescritical for receptor functions. CD16A is a low affinity FcγR withlimited capacity to engage therapeutic mAb-coated target cells. CD16Aalso undergoes a rapid downregulation in expression upon cell activationthat markedly reduces its cell surface density and avidity for IgG.CD16A downregulation occurs by a proteolytic event at an extracellularsite proximal to the plasma membrane and is referred to as ectodomainshedding. The location of this cleavage site has been reported (Jing etal., 2015. PLoS One 10:e0121788) and is shown schematically in FIG. 2and FIG. 13A.

CD64 (FcγRI) is another IgG Fc receptor, and is expressed by monocytes,macrophages, and activated neutrophils. CD64 is a high affinity IgGreceptor. This receptor does not undergo ectodomain shedding upon cellactivation and does not naturally transduce signals for ADCC in NKcells.

This disclosure describes a chimeric FcγR that includes a CD16A domainand a CD64 domain. The chimeric receptor includes the extracellularregion of human CD64 and the cytoplasmic region of human CD16A, anexemplary embodiment of which is shown schematically in FIG. 2 and FIG.13A as CD64/16A. In various embodiments, the chimeric CD64/CD16Areceptor can include the CD64 transmembrane region or the CD16Atransmembrane region. The CD64/16A construct has been engineered so thatit lacks the CD16A extracellular cleavage site and thus is notsusceptible to ectodomain shedding (FIG. 2; FIG. 13A), but includes atleast a portion of the CD16A intracellular region that is involved inintracellular signaling.

Also, while described herein in the context of an exemplary embodimentin which the CD64 domain and the CD16A contain amino acid sequences ofhuman CD64 and human CD16A, respectively, the chimeric FcγR describedherein can include an amino acid sequence that is, or is derived from,any suitable CD64 or CD16A natively expressed by any species. FIG. 18and FIG. 19 provide amino acid sequence alignments of human and canineamino acid sequences for CD16A (FIG. 18) and CD64 (FIG. 19).

As used herein, the amino acid sequence of a domain is “derived from” athe amino acid sequence of a reference polypeptide if the amino acidsequence of the domain possesses a specified amount of sequencesimilarity and/or sequence identity compared to the amino acid sequenceof the reference polypeptide. Sequence similarity of can be determinedby aligning the residues of the two polypeptides (for example, thedomain amino acid sequence and the amino acid sequence of the referenceCD16A or CD64 polypeptide) to optimize the number of identical aminoacids along the lengths of their sequences. Gaps in either or bothsequences are permitted in making the alignment in order to optimize thenumber of identical amino acids, although the amino acids in eachsequence must nonetheless remain in their proper order.

A pair-wise comparison analysis of amino acid sequences can be carriedout using the BESTFIT algorithm in the GCG package (version 10.2,Madison Wis.). Alternatively, polypeptides may be compared using theBlastp program of the BLAST 2 search algorithm, as described by Tatianaet al., (FEMS Microbiol Lett, 174, 247-250 (1999)), and available on theNational Center for Biotechnology Information (NCBI) website. Thedefault values for all BLAST 2 search parameters may be used, includingmatrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gapx_dropoff=50, expect=10, wordsize=3, and filter on.

The amino acid sequence of a domain is “derived from” a the amino acidsequence of a reference polypeptide if the amino acid sequence of thedomain possesses a specified degree of amino acid sequence “identity” oramino acid sequence “similarity.” Amino acid sequence identity refers tothe presence of identical amino acids. Amino acid sequence similarityrefers to the presence of not only identical amino acids but also thepresence of conservative substitutions. A conservative substitution foran amino acid may be selected from other members of the class to whichthe substituted amino acid belongs. For example, it is well-known in theart of protein biochemistry that an amino acid belonging to a groupingof amino acids having a particular size or characteristic (such ascharge, hydrophobicity and hydrophilicity) can be substituted foranother amino acid without altering the activity of a protein,particularly in regions of the protein that are not directly associatedwith biological activity. For example, nonpolar (hydrophobic) aminoacids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan, and tyrosine. Polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. Conservative substitutionsinclude, for example, Lys for Arg and vice versa to maintain a positivecharge; Glu for Asp and vice versa to maintain a negative charge; Serfor Thr so that a free —OH is maintained; and Gln for Asn to maintain afree —NH₂. Likewise, biologically active analogs of a polypeptidecontaining deletions or additions of one or more contiguous ornoncontiguous amino acids that do not eliminate a functional activity ofthe polypeptide are also contemplated.

The amino acid sequence of a CD16A domain or a CD64 domain is “derivedfrom” a reference amino acid sequence if the domain amino acid sequencehas at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence similarity to the reference amino acidsequence.

The amino acid sequence of a CD16A domain or a CD64 domain is “derivedfrom” a reference amino acid sequence if the domain amino acid sequencehas at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity to the reference amino acidsequence.

For the purpose of determining whether a domain amino acid sequence is“derived from” a specified reference amino acid sequence, exemplarysuitable reference polypeptides include human CD16A (SEQ ID NO:6),canine CD16A (SEQ ID NO:5), human CD64 (SEQ ID NO:8), canine CD64 (SEQID NO:7), canine CD64sp (SEQ ID NO:25), or the corresponding domains ofany of the constructs listed in Table 1.

Also, while described herein in the context of an exemplary embodiment,illustrated in FIG. 2 and FIG. 13A, the chimeric receptor may bedesigned to include a different fusion points between CD64 and CD16Athat that shown in FIG. 2 and FIG. 13A, to include the CD16A cleavageregion, modified functional motifs in regions of CD64 or CD16A, and/oradding an additional signaling domain, such as, for example, a signalingdomain of CD27, CD28, CD134 (OX40), CD137 (4-1BB), FcRγ, or CD3. Thechimeric receptor may therefore be designed to increase theproliferation of NK cells or other effector cells, increase survival ofNK cells or other effector cells, increase potency of NK cells or othereffector cells, and/or decrease NK cell exhaustion in vivo.

In certain embodiments, the chimeric FcγR can be modified to include acytoplasmic domain or a signaling domain that confers additionalfunctionality to the chimeric receptor. For example, a chimeric FcγR caninclude a functional portion of CD28, which transduces signals involvedin T-cell proliferation, survival, and cytokine production. As anotherexample, a chimeric FcγR can include a functional portion of 4-1BB,which contributes to the clonal expansion, survival, and development ofhematopoietic cells. As another example, a chimeric FcγR can include afunctional portion of the CD3ζ cytoplasmic domain, which contains threeimmunoreceptor tyrosine-based activation motifs (ITAMs) that triggerintracellular signal-transduction pathways for ADCC, cytokineproduction, and cell proliferation and survival. As another example, achimeric FcγR can include a functional portion of the FcRγ cytoplasmicdomain, which contains an ITAM that preferentially recruits Syk kinaseto mediate intracellular signals for ADCC, cytokine production, and cellproliferation and survival. As another example, a chimeric FcγR caninclude a functional portion of the DAP10 cytoplasmic domain, whichcontains a YxxM motif that specifically activates phosphatidylinositol3-kinase-dependent signaling pathways for cytotoxicity, cell survival,and proliferation of NK and T cells. As another example, a chimeric FcγRcan include a functional portion of the DAP12 cytoplasmic domain, whichcontains an ITAM that triggers signals for cytotoxicity, survival, andproliferation of NK cells and T cells. As another example, a chimericFcγR can include a functional portion of the NKG2D (or CD314)transmembrane domain, which specifically associates with DAP10 tomediate signaling pathways for cytotoxicity, cell survival, andproliferation of NK and T cells. As another example, a chimeric FcγR caninclude a functional portion of the 2B4 (NKR2B4 or CD244) cytoplasmicdomain, which transduces signals of cytolytic granule polarizationinvolved in enhanced cytotoxicity of NK cells. As another example, achimeric FcγR can include a functional portion of the high affinity IgEreceptor FcεR transmembrane and cytoplasmic domains, which areconstitutively associated with its β-subunit and FcR γ-chain (FcRγ) tomediate the most potent degranulation signals in myeloid cells thatinitiates the allergic responses, which could be exploited for cancertherapy with the recombinant high affinity IgG Fc receptors.

Exemplary constructs that include exemplary cytoplasmic domain and/orsignaling domain modifications are listed in Table 1.

TABLE 1 Code Domains (EC/TM/CY/SDs)* Comments SEQ ID NO: rCD64#164/64/64/— 9 rCD64#2 64/64/mut64/— CD64 cytoplasmic domain 10 mutationrCD64#3 64/16A/16A/— 11 rCD64#4 64/16A/16A/28-BB-CD3ç 12 rCD64#564/16A/16A/28-BB-FcRγ 13 rCD64#6 64/16A/16A/28-BB-Dap10 14 rCD64#764/16A/16A/28-BB-Dap12 15 rCD64#8 64/28/28/BB-CD3ç 16 rCD64#964/FcεR/FcεR/— 17 rCD64#10 64/16A/mut16A/— Mutation on PKC 18phosphorylation site rCD64#11 64/G2D/2B4/CD3ç 19 rCD64#1264/16A/16A/2B4-CD3ç 20 rCD64#13 64/16A/16A/2B4-FcRγ 21 rCD64#1464/16A/16A/2B4-Dap10 22 rCD64#15 64/16A/16A/2B4-Dap12 23 rCD64#1664/64/64/2B4-CD3ç 24 *EC: Extracellular domain; TM: Transmembranedomain; CY: Cytoplasmic domain; SD: Signaling domain. 64: CD64, Highaffinity IgG Fc receptor FcγRI; mutCD64: High affinity IgG Fc receptorFcγR1 with cytoplasmic mutation that results in higher levels ofcytokine production and degranulation; 16A: C16A, Low affinity IgG Fcreceptor FcγRIIIA; —: no signaling domain; 28: CD28, a co-stimulatoryreceptor for cell proliferation and activation; BB: 4.1BB or CD137;CD3ç: CD3 ç-chain or CD247; FcRγ: FcR γ-chain; D10: DAP10 signalingadaptor; D12: DAP12 signaling adaptor; FceR: High affinity IgE Fcreceptor; 16-PKC⁻: CD16A cytoplasmic domain with mutations on PKCphosphorylation site to disrupt cytokine productions mediated by CD16A;G2D: NKG2D or CD314; 2B4: NKR2B4 or CD244.

The CD64/16A chimeric receptor can be encoded by a cDNA that can betranscribed and translated from an expression vector introduced into ahost cell to produce a recombinant cell. The host cell can include asuitable leukocyte-like cell or a primary leukocyte. Suitableleukocyte-like cells include, but are not limited to, a hematopoieticcell line or an induced pluripotent stem cell. Suitable primaryleukocytes include, but are not limited to, NK cells, monocytes,macrophages, neutrophils, or T lymphocytes. The expression of CD64/16Aby genetically-engineered leukocytes can increase the recombinant cell'seffector function in killing target cells—e.g., tumor cells andvirus-infected cells—in the presence of natural and therapeuticantibodies compared to the unmodified host cell. Because the CD64/16Achimeric receptor binds to IgG with high affinity, it may also befeasible to attach therapeutic mAbs to effector cells expressing theconstruct prior to their administration into patients. Hence, CD64/16Awith an attached therapeutic mAb would provide the effector cells with atargeting element to direct them to cancer locations.

NK92 cells are a human NK cell line that lacks expression of endogenousCD16A. NK cells were generated that express in a stable manner wildtypeCD16A, wildtype CD64, or the exemplary chimeric receptor CD64/16A. NK92cells expressing CD64/16A could be stained with an anti-CD64 mAb, butnot with an anti-CD16 mAb (FIG. 3A). NK92-CD16A cells could be stainedwith an anti-CD16 mAb, but not with an anti-CD64 mAb (FIG. 3B). NK92cells expressing CD64 could be stained with an anti-CD64 mAb, but not byand isotype-matched negative control mAb (FIG. 20A).

FIG. 3C shows the ability of NK92 cells expressing CD64/16A or CD16A tobind trastuzumab, a therapeutic mAb specific to HER2/EGFR2 overexpressedby certain malignancies. Non-transduced NK92 cells, NK92-CD64/16A cells,and NK92-CD16A cells were incubated with trastuzumab (5 μg/ml) for twohours at room temperature, washed to remove unbound antibody, incubatedwith an anti-human IgG second stage antibody conjugated to afluorophore, and then examined by flow cytometry. NK92-hCD64/16A cellsbound much higher levels of trastuzumab than did NK92-CD16A cells andNK92 cells.

FIG. 4 presents data that shows the exemplary chimeric receptor CD64/16Aconferred NK92 cells with an ADCC effector function. NK92 cellsexpressing CD64/16A or wildtype CD16A were incubated with the humanovarian cancer cell line SKOV-3 (20:1 ratio) in the presence or absenceof trastuzumab at various concentrations (0.005 μg/ml, 0.05 μg/ml. 0.5μg/ml, or 5 μg/ml). NK92 cells expressing either CD64/16A or wildtypeCD16A demonstrated SKOV-3 cytotoxicity in the presence of trastuzumab.NK92 cells expressing CD64/16A had higher levels of target cell killingthan did NK92-CD16A cells at all trastuzumab concentrations examined(FIG. 4A). NK92 cells expressing either CD64 also demonstrated SKOV-3cytotoxicity in the presence of trastuzumab (FIG. 20B). In addition,NK92-CD64/16A and NK92-CD16A cells were pretreated with trastuzumab at 5μg/ml or 10 μg/ml for two hours, washed to remove unbound antibody, andthen incubated with SKOV-3 cells. In this assay, NK92-CD64/16A cellsdemonstrated a marked enhancement in target cell killing when comparedto NK92-CD16A cells (FIG. 4B).

CD64/16A also was expressed in iPSCs and these cells were thendifferentiated into NK cells (referred to here as iNK cells). As shownin FIG. 5, iNK cells transduced with either CD64/16A or the empty vector(pKT2) as a control were compared for their expression of several NKcell markers. iNK-CD64/16A and iNK-pKT2 cells are CD56⁺ and CD3⁻,indicating that they are indeed NK cells. They also expressed similarlevels of various NK cell markers. iNK-CD64/16A and iNK-pKT2 cells werefound to express similar levels of CD16A, whereas only iNK-CD64/16A werestained by an anti-CD64 mAb (FIG. 5).

iNK-CD64/16A and iNK-pKT2 cells were evaluated for ADCC, as describedabove for the NK92 cells. iNK-CD64/16A and iNK-pKT2 cells were incubatedwith SKOV-3 cells at 10:1 ratio in the presence or absence oftrastuzumab (5 μg/ml). iNK-CD64/16A cells demonstrated increased SKOV-3cytotoxicity in the presence of trastuzumab and higher levels of ADCCwhen compared to iNK-pKT2 cells (FIG. 6).

Although the iNK-CD64/16A and iNK-pKT2 cells expressed similar levels ofCD16A (FIG. 5), the function blocking anti-CD16A mAb 3G8 only blockedADCC by the iNK-pKT2 cells (FIG. 6). Conversely, the function blockinganti-CD64 mAb 10.1 only blocked ADCC by the iNK-CD64/16A cells (FIG. 6).iNK-CD64/16A and iNK-pKT2 cells were also pretreated with trastuzumab at10 μg/ml for two hours, washed to remove unbound antibody, and thenincubated with SKOV-3 cells. In this assay, iNK-CD64/16A cellsdemonstrated a distinct enhancement in target cell killing when comparedto iNK-pKT2 cells (FIG. 7). MA148 is a human ovarian cancer cell linethat expresses considerably lower levels of HER2 when compared to SKOV-3cells. ADCC was evaluated in iNK-CD64/16A and iNK-pKT2 cells whenexposed to MA148 cells at various ratios in the presence or absence oftrastuzumab (5 μg/ml). Again, iNK-CD64/16A cells demonstratedsignificantly higher levels of tumor cell killing than did iNK-pKT2cells at all effector to target cell ratios when in the presence oftrastuzumab (FIG. 8).

The chimeric receptor CD64/16A (FIG. 2; FIG. 13A) was stably expressedin the human NK cell line NK92. These cells lack endogenous FcγRs buttransduced cells expressing exogenous CD16A can mediate ADCC. As shownin FIG. 13B, an anti-CD64 mAb stained NK92 cells expressing CD64/16Acells, but not parent NK92 cells or NK92 cells expressing CD16A. Ananti-CD16 mAb stained NK92 cells expressing CD16A, but not NK92 cellsexpressing CD64/16A or parent NK92 cells (FIG. 13B). CD16A undergoesectodomain shedding by ADAM17 upon NK cell activation resulting in itsrapid downregulation in expression. CD16A and its isoform CD16B onneutrophils is cleaved by ADAM17, and this occurs at an extracellularregion proximal to the cell membrane. The ADAM17 cleavage region ofCD16A is not present in CD64 or CD64/16A (FIG. 13A). CD16A underwenta >50% decrease in expression upon NK92 stimulation by ADCC, whereasCD64/16A demonstrated little to no downregulation (FIG. 13C).

To evaluate the function of CD64/16A, the ability of CD64/16A toinitiate E:T conjugation, induce ADCC, and stimulate cytokine productionupon NK cell engagement of antibody-bound tumor cells was examined.Prior to the release of its granule contents, an NK cell forms a stableconjugate with a target cell. NK92-CD64/16A cell and SKOV-3 cellconjugation were examined using a two-color flow cytometric approach.SKOV-3 cells are an ovarian cancer cell line that express HER2, and thisassay was performed in the absence and presence of the anti-HER2therapeutic mAb trastuzumab. The bicistronic vector containing CD64/16Aalso expressed eGFP and its fluorescence was used to identify the NK92cells. SKOV-3 cells were labeled with the fluorescent dye CellTraceViolet. E:T conjugation resulted in two-color events that were evaluatedby flow cytometry. Incubating NK92-CD64/16A cells with SKOV-3 cellsresulted in a very low level of conjugation after initial exposure thatincreased after 60 minutes of exposure (FIG. 14A). However, in thepresence of trastuzumab, NK92-CD64/16A cell and SKOV-3 conjugation wasappreciably enhanced (FIG. 14A). This increase in conjugationcorresponded with higher levels of target cell killing. As shown in FIG.14B, SKOV-3 cell cytotoxicity by NK92-CD64/16A cells varied depending onthe trastuzumab concentration and E:T ratio. To confirm the role ofCD64/16A in the induction of target cell killing, the ADCC assay wasperformed in the presence and absence of the anti-CD64 mAb 10.1 (FIG.14C), which blocks IgG binding. Cytokine production is also inducedduring ADCC and NK cells are major producers of IFNγ. NK92-CD64/16Acells exposed to SKOV-3 cells and trastuzumab produced considerablyhigher levels of IFNγ than when exposed to SKOV-3 cells alone (FIG.14D). Taken together, the above findings demonstrate that the CD64component of the recombinant receptor engages tumor-bound antibody, andthat the CD16A component promotes intracellular signaling leading todegranulation and cytokine production.

CD64 is distinguished from the other FcγR members by its unique thirdextracellular domain, which contributes to its high affinity and stablebinding to soluble monomeric IgG. NK92 cells expressing CD64/16A or theCD16A-176V higher affinity variant were compared for their ability tocapture soluble therapeutic mAbs. The NK92 cell transductants examinedexpressed similar levels of CD64/16A and CD16A (FIG. 15A). NK92 celltransductants were incubated with trastuzumab for two hours, excessantibody was washed away, stained with a fluorophore-conjugatedanti-human IgG antibody, and then evaluated by flow cytometry. As shownin FIG. 15B, NK92-CD64/16A cells captured considerably higher levels oftrastuzumab than did the NK92-CD16A cells (8.1 fold increase±1.3,mean±SD of three independent experiments). Moreover, the NK92-CD64/16Acells efficiently captured the tumor-targeting mAbs cetuximab andrituximab, as well as the fusion protein L-selectin/Fc (FIG. 15C).

NK92-CD64/16A cells with a captured tumor-targeting mAb were tested todetermine whether the cells mediated ADCC. For this assay, equal numbersof NK92-CD64/16A and NK92-CD16A cells were incubated with the sameconcentration of soluble trastuzumab, washed, and exposed to SKOV-3cells. Target cell killing by NK92-CD64/16A cells with capturedtrastuzumab was significantly higher than NK92-CD64/16A cells alone, andwas superior to NK92-CD16A cells treated with or without trastuzumab atall E:T ratios examined (FIG. 15D). In contrast, SKOV-3 cytotoxicity byNK92-CD16A and NK92-CD64/16A cells was not significantly different whentrastuzumab was present and not washed out (FIG. 15E), thusdemonstrating equivalent cytotoxicity by both transductants. Takentogether, these findings show that NK92 cells expressing CD64/16A canstably bind soluble anti-tumor mAbs and IgG fusion proteins, and thatthese can serve as targeting elements to kill cancer cells.

Expression and Function of CD64/16A in iPSC-Derived NK Cells

Undifferentiated iPSCs were transduced to express CD64/16A using aSleeping Beauty transposon plasmid for nonrandom gene insertion andstable expression. iPSCs were differentiated into hematopoietic cellsand then iNK cells as described in the EXAMPLE 2. CD34⁺CD43⁺CD45⁺ cellswere generated, further differentiated into iNK cells, and these cellswere expanded for analysis using recombinant IL-2 and aAPCs. CD56⁺CD3⁻is a hallmark phenotype of human NK cells, and these cells composed themajority of our differentiated cell population (FIG. 16). Expression ofactivating and inhibitory receptors on the iNK cells also were assessedand compared to expression by peripheral blood NK cells. Certainreceptors, such as CD16A, were expressed by similar proportions of thetwo NK cell populations. The expanded iNK cells, however, lackedexpression of the inhibitory KIR receptors KIR2DL2/3, KIR2DL1, andKIR3DL1 and also certain activating receptors (NKp46 and NKG2D) (FIG.16). Another difference compared to peripheral blood NK cells was thatthe iNK cells were stained with an anti-CD64 mAb (FIG. 16),demonstrating the expression of CD64/16A.

To assess the function of CD64/16A in iNK cells, iNK cells derived fromiPSCs transduced with either an pKT2 empty vector or pKT2-CD64/16A werecompared. The NK cell markers mentioned above were expressed at similarlevels and proportions by two iNK cell populations (data not shown),including CD16A (FIG. 17A), but only iNK-CD64/16A cells were stained byan anti-CD64 mAb (FIG. 17A). Both iNK tranductants demonstratedincreased SKOV-3 cell killing when in the presence of trastuzumab, yetiNK-CD64/16A cells mediated significantly higher levels of ADCC than didthe iNK-pKT2 control cells (FIG. 17B). The anti-CD16 function blockingmAb 3G8, but not the anti-CD64 mAb 10.1, effectively inhibited ADCC bythe iNK-pKT2 cells (FIG. 17B). Conversely, 10.1, but not 3G8, blockedADCC by the iNK-CD64/16A cells (FIG. 17B). These findings show that theiNK cells were cytolytic effectors responsive to CD16A and CD64/16Aengagement of antibody-bound tumor cells.

Also, iNK-CD64/16A and iNK-pKT2 cells were treated with solubletrastuzumab, excess antibody was washed away, and the cells were exposedto SKOV-3 cells. Under these conditions, ADCC by the iNK-CD64/16A cellswas strikingly higher than the iNK-pKT2 cells (FIG. 17C), furtherestablishing that CD64/16A can capture soluble anti-tumor mAbs thatserve as a targeting element for tumor cell killing.

Taken together, the data show that CD64/16A binds therapeutic mAbs withhigher affinity than CD16A. Moreover, CD64/16A expressed in NK92 cellsand iNK cells confers cells with the ability to mediate higher levels ofADCC than NK92 cells and iNK cells expressing wildtype or endogenousCD16A, respectively. NK cells expressing CD64/16A facilitated cellconjugation with antibody-bound tumor cells, cytotoxicity, and IFNγproduction, demonstrating function by both components of the recombinantFcγR. NK92 cells and iNK cells expressing CD64/16A can be pre-loadedwith a therapeutic mAb prior to their exposure to target cells. Cellsexpressing the chimeric receptor and preloaded with therapeutic antibodymay allow one to modify engineered NK cells with different therapeuticmAbs for targeting elements of multiple types of cancer. Finally,CD64/16A lacks the ADAM17 cleavage region found in CD16A and it did notundergo the same level of downregulation in expression during ADCC.

CD64/16A was shown to be functional in two NK cell platforms, the NK92cell line and primary NK cells derived from iPSCs. NK92 cells lackinhibitory KIR receptors and show high levels of natural cytotoxicitycompared to other NK cell lines derived from patients. NK92 cells havebeen broadly used to express modified genes to direct their cytolyticeffector function, have been evaluated in preclinical studies, and areundergoing clinical testing in cancer patients. iPSCs are also veryamendable to genetic engineering and can be differentiated into NK cellsexpressing various receptors to direct their effector functions. The iNKcells generated in this study lacked several inhibitory and activatingreceptors that are indicators of an immature state. In someapplications, therapeutic iNK cells lacking inhibitory receptors andcertain activating receptors may allow for more directed and/oreffective tumor cell killing by engineered receptors.

The iNK cells expressed endogenous CD16A and mediated ADCC, thus theywere cytotoxic effector cells. An individual NK cell can kill multipletumor cells in different manners. This includes by a process ofsequential contacts and degranulations, referred to as serial killing,and by the localized dispersion of its granule contents that killssurrounding tumor cells, referred to as bystander killing. InhibitingCD16A shedding has been reported to slow NK cell detachment from targetcells and reduce serial killing by NK cells in vitro. Due to the CD64component and its lack of ectodomain shedding, NK cells expressingCD64/16A could be less efficient at serial killing and more efficient atbystander killing.

NK cells expressing CD64/16A have several potential advantages as atherapeutic in combination with a therapeutic antibody. Modifying NKcells expressing CD64/16A with an antibody can reduce the dosage oftherapeutic antibodies administered to patients. Fusion proteinscontaining a human IgG Fc region, such as L-selectin/Fc, also can becaptured by CD64/16A and this may provide further options for directingthe tissue and tumor antigen targeting of engineered NK cells. The NK92and iNK cell platforms for adoptive cell therapies also can be readilygenetically modified on a clonal level and expanded intoclinical-scalable cell numbers to produce engineered NK cells withimproved effector activities as an off-the-shelf therapeutic for cancerimmunotherapy.

In some embodiments, iPSC-derived NK cells that express CD64/16A canexhibit increased in vivo anti-cancer activity in the presence oftherapeutic mAbs. For example, NOD/SCID/γc^(−/−) (NSG) mice and humancancer cell lines that are stably engineered to express fireflyluciferase can be used for bioluminescent imaging to test iNK cellactivity against cancer cells. The SKOV-3 and MA148 ovarian cancer cellline cans serve as model in vivo targets. NSG female mice can besubjected to sublethal irradiation, then injected intraperitoneally withtumor cells for bioluminescent imaging to quantify tumor growth orregression. Mice are given IL-2 and/or IL-15 every other day for fourweeks to promote in vivo survival of the NK cells. Therapeutic antibody(e.g., trastuzumab) can be administered—e.g., once weekly for fourweeks. Tumor growth/regression can be monitored by, for example,bioluminescent imaging and weighing the mice. Mice also can be bled(e.g., weekly) to quantify human NK cell survival, and one can furtherevaluate the expression/cell surface levels of various effector functionmarkers by FACS. Evidence of metastasis can be determined by, forexample, harvesting internal organs and examining the internal organsfor metastasis.

In some embodiments, the number of cells in the therapeutic compositionis at least 0.1×10⁵ cells, at least 1×10⁵ cells, at least 5×10⁵ cells,at least 1×10⁶ cells, at least 5×10⁶ cells, at least 1×10⁷ cells, atleast 5×10⁷ cells, at least 1×10⁸ cells, at least 5×10⁸ cells, at least1×10⁹ cells, or at least 5×10⁹ cells, per dose. In some embodiments, thenumber of cells in the therapeutic composition is about 0.1×10⁵ cells toabout 1×10⁶ cells, per dose; about 0.5×10⁶ cells to about 1×10⁷ cells,per dose; about 0.5×10⁷ cells to about 1×10⁸ cells, per dose; about0.5×10⁸ cells to about 1×10⁹ cells, per dose; about 1×10⁹ cells to about5×10⁹ cells, per dose; about 0.5×10⁹ cells to about 8×10⁹ cells, perdose; about 3×10⁹ cells to about 3×10¹⁰ cells, per dose, or any rangein-between. Generally, 1×10⁸ cells/dose translates to 1.67×10⁶ cells/kgfor a 60 kg patient.

In one embodiment, the number of cells in the therapeutic composition isthe number of immune cells in a partial or single cord of blood, or isat least 0.1×10⁵ cells/kg of bodyweight, at least 0.5×10⁵ cells/kg ofbodyweight, at least 1×10⁵ cells/kg of bodyweight, at least 5×10⁵cells/kg of bodyweight, at least 10×10⁵ cells/kg of bodyweight, at least0.75×10⁶ cells/kg of bodyweight, at least 1.25×10⁶ cells/kg ofbodyweight, at least 1.5×10⁶ cells/kg of bodyweight, at least 1.75×10⁶cells/kg of bodyweight, at least 2×10⁶ cells/kg of bodyweight, at least2.5×10⁶ cells/kg of bodyweight, at least 3×10⁶ cells/kg of bodyweight,at least 4×10⁶ cells/kg of bodyweight, at least 5×10⁶ cells/kg ofbodyweight, at least 10×10⁶ cells/kg of bodyweight, at least 15×10⁶cells/kg of bodyweight, at least 20×10⁶ cells/kg of bodyweight, at least25×10⁶ cells/kg of bodyweight, at least 30×10⁶ cells/kg of bodyweight,1×10⁸ cells/kg of bodyweight, 5×10⁸ cells/kg of bodyweight, or 1×10⁹cells/kg of bodyweight.

In one embodiment, a dose of cells is delivered to a subject. In oneillustrative embodiment, the effective amount of cells provided to asubject is at least 2×10⁶ cells/kg, at least 3×10⁶ cells/kg, at least4×10⁶ cells/kg, at least 5×10⁶ cells/kg, at least 6×10⁶ cells/kg, atleast 7×10⁶ cells/kg, at least 8×10⁶ cells/kg, at least 9×10⁶ cells/kg,or at least 10×10⁶ cells/kg, or more cells/kg, including all interveningdoses of cells.

In another illustrative embodiment, the effective amount of cellsprovided to a subject is about 2×10⁶ cells/kg, about 3×10⁶ cells/kg,about 4×10⁶ cells/kg, about 5×10⁶ cells/kg, about 6×10⁶ cells/kg, about7×10⁶ cells/kg, about 8×10⁶ cells/kg, about 9×10⁶ cells/kg, or about10×10⁶ cells/kg, or more cells/kg, including all intervening doses ofcells.

In another illustrative embodiment, the effective amount of cellsprovided to a subject is from about 2×10⁶ cells/kg to about 10×10⁶cells/kg, about 3×10⁶ cells/kg to about 10×10⁶ cells/kg, about 4×10⁶cells/kg to about 10×10⁶ cells/kg, about 5×10⁶ cells/kg to about 10×10⁶cells/kg, 2×10⁶ cells/kg to about 6×10⁶ cells/kg, 2×10⁶ cells/kg toabout 7×10⁶ cells/kg, 2×10⁶ cells/kg to about 8×10⁶ cells/kg, 3×10⁶cells/kg to about 6×10⁶ cells/kg, 3×10⁶ cells/kg to about 7×10⁶cells/kg, 3×10⁶ cells/kg to about 8×10⁶ cells/kg, 4×10⁶ cells/kg toabout 6×10⁶ cells/kg, 4×10⁶ cells/kg to about 7×10⁶ cells/kg, 4×10⁶cells/kg to about 8×10⁶ cells/kg, 5×10⁶ cells/kg to about 6×10⁶cells/kg, 5×10⁶ cells/kg to about 7×10⁶ cells/kg, 5×10⁶ cells/kg toabout 8×10⁶ cells/kg, or 6×10⁶ cells/kg to about 8×10⁶ cells/kg,including all intervening doses of cells.

Some variation in dosage will necessarily occur depending on thecondition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject.

In some embodiments, the therapeutic use of cells is a single-dosetreatment. In some embodiments, the therapeutic use of derivedhematopoietic lineage cells is a multi-dose treatment. In someembodiments, the multi-dose treatment is one dose every day, every 3days, every 7 days, every 10 days, every 15 days, every 20 days, every25 days, every 30 days, every 35 days, every 40 days, every 45 days, orevery 50 days, or any number of days in-between.

A compositions that includes a population of cells described herein canbe sterile, and can be suitable and ready for administration (i.e., canbe administered without any further processing) to human patients. Acell-based composition that is ready for administration means that thecomposition does not require any further processing or manipulationprior to transplant or administration to a subject. In some embodiments,such a population of cells can include an isolated population of cellsthat are expanded and/or modulated prior to administration with one ormore agents.

In certain embodiments, the primary stimulatory signal and theco-stimulatory signal for the therapeutic cells can be provided bydifferent protocols. For example, the agents providing each signal canbe in solution or coupled to a surface. When coupled to a surface, theagents can be coupled to the same surface (i.e., in “cis” formation) orto separate surfaces (i.e., in “trans” formation). Alternatively, oneagent can be coupled to a surface and the other agent in solution. Inone embodiment, the agent providing the co-stimulatory signal can bebound to a cell surface and the agent providing the primary activationsignal is in solution or coupled to a surface. In certain embodiments,both agents can be in solution. In another embodiment, the agents can bein soluble form, and then cross-linked to a surface, such as a cellexpressing Fc receptors or an antibody or other binding agent that willbind to the agents such as disclosed in U.S. Patent ApplicationPublication Nos. 20040101519 and 20060034810 for artificial antigenpresenting cells (aAPCs) that are contemplated for use in activating andexpanding T lymphocytes.

The therapeutic compositions suitable for administration to a patientcan include one or more pharmaceutically acceptable carriers (additives)and/or diluents (e.g., pharmaceutically acceptable medium, for example,cell culture medium), or other pharmaceutically acceptable components.Pharmaceutically acceptable carriers and/or diluents are determined inpart by the particular composition being administered, as well as by theparticular method used to administer the therapeutic composition.Accordingly, there is a wide variety of suitable formulations oftherapeutic compositions (see, e.g., Remington's PharmaceuticalSciences, 17^(th) ed. 1985, the disclosure of which is herebyincorporated by reference in its entirety) well known to those of skillin the art.

In particular embodiments, therapeutic cell compositions having anisolated population the cells as described herein also have apharmaceutically acceptable cell culture medium, or pharmaceuticallyacceptable carriers and/or diluents. A therapeutic compositioncomprising a population of the cells as disclosed herein can beadministered separately by intravenous, intraperitoneal, enteral, ortracheal administration methods or in combination with other suitablecompounds to affect the desired treatment goals.

These pharmaceutically acceptable carriers and/or diluents can bepresent in amounts sufficient to maintain a pH of the therapeuticcomposition of between about 3 and about 10. As such, the bufferingagent can be as much as about 5% on a weight to weight basis of thetotal composition. Electrolytes such as, but not limited to, sodiumchloride and potassium chloride can also be included in the therapeuticcomposition. In one aspect, the pH of the therapeutic composition is inthe range from about 4 to about 10. Alternatively, the pH of thetherapeutic composition is in the range from about 5 to about 9, fromabout 6 to about 9, or from about 6.5 to about 8. In another embodiment,the therapeutic composition includes a buffer having a pH in one of saidpH ranges. In another embodiment, the therapeutic composition has a pHof about 7. Alternatively, the therapeutic composition has a pH in arange from about 6.8 to about 7.4. In still another embodiment, thetherapeutic composition has a pH of about 7.4.

This disclosure also provides the use of a pharmaceutically acceptablecell culture medium in particular compositions and/or cultures of cellsas described herein. Such compositions are suitable for administrationto human subjects. Generally speaking, any medium that supports themaintenance, growth, and/or health of the iPSC-derived immune cells aresuitable for use as a pharmaceutical cell culture medium. In particularembodiments, the pharmaceutically acceptable cell culture medium is aserum free, and/or feeder-free medium. In various embodiments, theserum-free medium is animal-free, and can optionally be protein-free.Optionally, the medium can contain biopharmaceutically acceptablerecombinant proteins. Animal-free medium refers to medium wherein thecomponents are derived from non-animal sources. Recombinant proteinsreplace native animal proteins in animal-free medium and the nutrientsare obtained from synthetic, plant or microbial sources. Protein-freemedium, in contrast, is defined as substantially free of protein. Onehaving ordinary skill in the art would appreciate that the aboveexamples of media are illustrative and in no way limit the formulationof suitable media suitable and that there are many alternative suitablemedia known and available to those in the art.

In the preceding description and following claims, the term “and/or”means one or all of the listed elements or a combination of any two ormore of the listed elements; the terms “comprises,” “comprising,” andvariations thereof are to be construed as open ended—i.e., additionalelements or steps are optional and may or may not be present; unlessotherwise specified, “a,” “an,” “the,” and “at least one” are usedinterchangeably and mean one or more than one; and the recitations ofnumerical ranges by endpoints include all numbers subsumed within thatrange (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

In the preceding description, particular embodiments may be described inisolation for clarity. Unless otherwise expressly specified that thefeatures of a particular embodiment are incompatible with the featuresof another embodiment, certain embodiments can include a combination ofcompatible features described herein in connection with one or moreembodiments.

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1 Generation of Human CD64/16A Expression Constructs

Total RNA was isolated from peripheral blood leukocytes (PBL) usingTRIzol total RNA isolation reagent (Invitrogen, Thermo FisherScientific, Carlsbad, Calif.). Human PBL cDNA was synthesized with theSuperScript Preamplification System (Invitrogen, Thermo FisherScientific, Carlsbad, Calif.). The CD64/16A includes a human CD64extracellular domain (CD64-EC) and CD16A transmembrane and cytoplasmicdomains (CD16A-TM-CY). The chimeric construct was generated byoverlapping PCR to create EcoR I-flanked RT-PCR products of chimericcDNA. CD64-EC cDNA fragment (885 bps) was amplified from human PBL cDNAusing the forward primer 5′-CGG GAA TTC GGA GAC AAC ATG TGG TTC TTG ACAA-3′(SEQ ID NO:28) and reverse primer 5′-TTG GTA CCC AGG TGG AAA GAA GCCAAG CAC TTG AAG CTC CAA-3′; SEQ ID NO:29). CD16A-TM-CY cDNA fragment(195 bps) was amplified from human PBL cDNA using the forward primer5′-TTG GAG CTT CAA GTG CTT GGC TTC TTT CCA CCT GGG TAC CAA-3′ (SEQ IDNO:30) and reverse primer 5′-CCG GAA TTC TCA TTT GTC TTG AGG GTC CTTTCT-3′ (SEQ ID NO:31). The PCR fragments of CD64-EC cDNA and CD16A-TM-CYcDNA were purified with QIAquick gel extraction kit (Qiagen, Hilden,Germany) and mixed together with the forward primer 5′-CGG GAA TTC GGAGAC AAC ATG TGG TTC TTG ACA A-3′ (SEQ ID NO:32) and the reverse primer5′-CCG GAA TTC TCA TTT GTC TTG AGG GTC CTT TCT-3′ (SEQ ID NO:33). Forall primers listed above, underlined nucleotides are the EcoR I cuttingsite) to generate RT-PCR products for the chimeric receptor. RT-PCR wasperformed with Pfx50 DNA polymerase (Invitrogen, Thermo FisherScientific, Carlsbad, Calif.). The resultant CD64/CD16a cDNA wasinserted into retrovirus expression vector pBMN-IRES-EGFP (Addgene,Cambridge, Mass.) and pKT2 Sleeping Beauty transposon vector (Jing etal. 2015. PLoS One 10:e0121788; Hermanson et al. 2016. Stem Cells34:93-101). The sequences of all cloned constructs were confirmed bydirect sequencing from both directions on an ABI 377 sequencer with ABIBigDye terminator cycle sequencing kit (Applied Biosystems, ThermoFisher Scientific, Foster City, Calif.).

Stable Expression of CD64/16A in NK Cells

NK92 cells, a human NK cell line that is deficient for endogenous CD16Aexpression, were stably transduced with pBMN-IRES-EGFP retrovirusexpression constructs containing CD64/16A or wildtype CD16A cDNA usingretrovirus infection procedures described previously (Jing et al. 2015.PLoS One 10:e0121788). Human iPSCs (UCBiPS7, derived from umbilical cordblood CD34 cells) were stably transduced with the CD64/16A-pKT2expression construct using a Sleeping Beauty transposon system, aspreviously described (Jing et al. 2015. PLoS One 10:e0121788). Thetransduced iPSC cells were differentiated into hematopoietic cells thenmature NK cell as previously described (Jing et al. 2015. PLoS One10:e0121788). eGFP fluorescence and surface expressions of CD64, CD16,and various NK cell phenotypic markers were determine using flowcytometry analysis.

Example 2 Antibodies

All mAbs to human hematopoietic and leukocyte phenotypic markers aredescribed in Table 2. All isotype-matched negative control mAbs werepurchased from BioLegend (San Diego, Calif.). APC-conjugated F(ab′)₂donkey anti-human or goat anti-mouse IgG (H+L) were purchased fromJackson ImmunoResearch Laboratories (West Grove, Pa.). The human IgG1mAbs trastuzumab/Herceptin and rituximab/Rituxan, manufactured byGenentech (South San Francisco, Calif.), and cetuximab/Erbitux,manufactured by Bristol-Myers Squibb (Lawrence, N.J.), were purchasedthrough the University of Minnesota Boynton Pharmacy. Recombinant humanL-selectin/IgG1 Fc chimera was purchased from R&D Systems (Minneapolis,Minn.).

TABLE 2 Antibodies Antigen Clone Fluorophore Company CD56 HCD56 PE-CY7BioLegend, San Diego, CA CD3 HIT3a PE BioLegend CD16 3G8 APC BioLegendCD16 3G8 none Ancell, Bayport, MN CD7 CD7-6B7 PE/CY5 BioLegendCD336/NKp44 P44-8 APC BioLegend CD335/NKp46 9E2 APC BioLegendCD159a/NKG2A Z199 APC Beckman Coulter, Brea, CA CD314/NKG2D 1D11PerCP/Cy5.5 BioLegend CD158a/KIR2DL1 HP-MA4 PE BioLegendCD158b1/KIR2DL2/L3 DX27 PE BioLegend CD158e1/KIR3DL1 DX9 PE BioLegendCD94 DX22 PE BioLegend CD64  10.1 APC BioLegend CD64  10.1 none AncellCD34 561 PE BioLegend CD45 2D1 APC BioLegend CD43 CD43-10G7 APCBioLegend CD62L/L-selectin LAM1-116 APC Ancell

Generation of Human CD64/16A

Total RNA was isolated from human peripheral blood leukocytes usingTRIzol total RNA isolation reagent (Invitrogen, Carlsbad, Calif.) andcDNA was synthesized with the SuperScript preamplification system(Invitrogen, Carlsbad, Calif.). The recombinant CD64/16A is comprised ofhuman CD64 extracellular domain and CD16A transmembrane and cytoplasmicdomains. PCR fragments for CD64 (885 bps) and CD16A (195 bps) wereamplified from the generated cDNA. The PCR fragments were purified andmixed together with the forward primer 5′-CGG GAA TTC GGA GAC AAC ATGTGG TTC TTG ACA A-3′ (SEQ ID NO:28), the reverse primer 5′-CCG GAA TTCTCA TTT GTC TTG AGG GTC CTT TCT-3′ (SEQ ID NO: 31), and Pfx50 DNApolymerase (Invitrogen) to generate the recombinant CD64/16A receptor.For both primers, underlined nucleotides are EcoR I sites. CD64/CD16Aand CD16A cDNA (CD16A-176V variant) was inserted into the retroviralexpression vector pBMN-IRES-EGFP and virus was generated for NK92 celltransduction, as previously described (Jing et al., 2015. PLoS One10(3):e0121788). Additionally, CD64/CD16A cDNA was inserted into thepKT2 sleeping beauty transposon vector and used along with SB100Xtransposase for iPSC transduction, as previously described (Jing et al.,2015. PLoS One 10(3):e0121788). The nucleotide sequences of allconstructs were confirmed by direct sequencing from both directions onan ABI 377 sequencer with ABI BigDye terminator cycle sequencing kit(Applied Biosystems, Foster City, Calif.).

Cells

This study was carried out in accordance with and approved by theInstitutional Review Board at the University of Minnesota. All subjectsgave written informed consent in accordance with the Declaration ofHelsinki. Fresh human peripheral blood leukocytes from plateletpheresiswere obtained from Innovative Blood Resources (St. Paul, Minn.).Peripheral blood mononuclear cells were further enriched usingFicoll-Paque Plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) andNK cells were purified by negative depletion using an EasySep human NKcell kit (StemCell Technologies, Cambridge, Mass.), as per themanufacturer's instructions, with >95% viability and 90-95% enrichmentof CD56⁺CD3⁻ lymphocytes. Viable cell counting was performed using aCountess II automated cell counter (Life Technologies Corporation,Bothell, Wash.). The human NK cell line NK92 and the ovarian cancer cellline SKOV-3 were obtained from ATCC (Manassas, Va.) and cultured per themanufacturer's directions. The NK92 cells required IL-2 for growth (500IU/ml), which was obtained from R&D Systems (Minneapolis, Minn.). Forall assays described below, cells were used when in log growth phase.

The iPSCs UCBiPS7, derived from umbilical cord blood CD34 cells, havebeen previously characterized and were cultured and differentiated intohematopoietic progenitor cells as described with some modifications(Jing et al., 2015. PLoS One 10:e0121788; Ni et al., 2013. Methods inmolecular biology 1029:33-41; Knorr et al., 2013. Stem Cells Transl Med2(4):274-283; Ni et al., 2014. Stem Cells 32(4):1021-1031; Hermanson etal., 2016. Stem Cells 34(1):93-101). iPSCs culture and hematopoieticdifferentiation was performed using TeSR-E8 medium and a STEMdiffHematopoietic Kit (StemCell Technologies, Cambridge, Mass.), which didnot require the use of mouse embryonic fibroblast feeder cells, TrypLEadaption, spin embryoid body formation, or CD34⁺ cell enrichment. iPSCcells during passage were dissociated with Gentle Cell DissociationReagent (StemCell Technologies, Cambridge, Mass.), and iPSC aggregates≥50 μm in diameter were counted with a hemocytometer and diluted to20-40 aggregates/ml with TeSR-E8 medium. Each well of a 12-well platewas pre-coated with Matrigel Matrix (Corning Inc., Tewksbury, Mass.) andseeded with 40-80 aggregates in 2 ml of TeSR-E8 medium. Cell aggregateswere cultured for 24 hours before differentiation with the STEMdiffHematopoietic Kit (StemCell Technologies, Cambridge, Mass.) according tothe manufacturer's instructions. At day 12 of hematopoietic progenitorcell differentiation, the percentage of hematopoietic progenitor cellswas determined using flow cytometric analysis with anti-CD34, anti-CD45,and anti-CD43 mAbs. NK cell differentiation was performed as previouslydescribed (Woll et al., 2009. Blood 113(24):6094-6101). The iPSC-derivedNK cells (referred to here as iNK cells) were expanded for examinationusing γ-irradiated K562-mbIL21-41BBL feeder cells (1:2 ratio) in cellexpansion medium [60% DMEM, 30% Ham's F12, 10% human AB serum (ValleyBiomedical, Winchester, Va.), 20 μM 2-mercaptoethanol, 50 μMethanolamine, 20 μg/ml ascorbic Acid, 5 ng/ml sodium selenite, 10 mMHEPES, and 100-250 IU/ml IL-2 (R&D Systems)], as described previously(Jing et al., 2015. PLoS One 10:e0121788; Knorr et al., 2013. Stem CellsTransl Med 2(4):274-283; Ni et al., 2014. Stem Cells 32(4):1021-1031;Hermanson et al., 2016. Stem Cells 34(1):93-101).

Cell Staining, Flow Cytometry, and ELISA

For cell staining, nonspecific antibody binding sites were blocked andcells were stained with the indicated antibodies and examined by flowcytometry, as previously described (Romee et al, 2013. Blood121(18):3599-3608; Jing et al., 2015. PLoS One 10:e0121788; Mishra etal., 2018. Cancer Immunol Immunother 67(9):1407-1416). For controls,fluorescence minus one was used as well as appropriate isotype-matchedantibodies since the cells of interest expressed FcRs. An FSC-A/SSC-Aplot was used to set an electronic gate on leukocyte populations and anFSC-A/F SC-H plot was used to set an electronic gate on single cells. AZombie viability kit was used to assess live vs. dead cells, as per themanufacturer's instructions (BioLegend, San Diego, Calif.).

To assess the capture of soluble trastuzumab, rituximab, cetuximab, orL-selectin/Fc chimera, transduced NK cells were incubated with 5 μg/mlof antibody for two hours at 37° C. in MEM-α basal media (Thermo FisherScientific, Waltham, Mass.) supplemented with IL-2 (200 IU/ml), HEPES(10 mM), and 2-mercaptoethanol (0.1 mM), washed with MEM-α basal media,and then stained on ice for 30 minutes with a 1:200 dilution ofAPC-conjugated F(ab′)2 donkey anti-human IgG (H+L). To detectrecombinant human L-selectin/Fc binding, cells were stained with theanti-L-selectin mAb APC-conjugated Lam1-116.

To compare CD16A, CD64, and CD64/16A staining levels on NK92 cells, therespective transductants were stained with a saturating concentration ofunconjugated anti-CD16 (3G8) or anti-CD64 (10.1) mAbs (5 μg/ml), washedextensively with dPBS (USB Corporation, Cleveland, Ohio) containing 2%goat serum and 2 mM sodium azide, and then stained with APC-conjugatedF(ab′)2 goat anti-mouse IgG (H+L). This approach was used since directlyconjugated anti-CD16 and anti-CD64 mAbs can vary in their levels offluorophore labeling. ELISA was performed by a cytometric bead-basedFlex Set assay for human IFNγ (BD Biosciences, San Jose, Calif.) per themanufacturer's instructions. All flow cytometric analyses were performedon a FACSCelesta instrument (BD Biosciences). Data was analyzed usingFACSDIVA v8 (BD Biosciences) and FlowJo v10 (Ashland, Oreg.).

Cell-Cell Conjugation Assay and ADCC

The pBMN-IRES-EGFP vector used to express CD64/16A in NK92 cells alsoexpresses eGFP. These cells were incubated for two hours at 37° C. inMEM-α basal media (Thermo Fisher Scientific, Waltham, Mass.)supplemented with IL-2 (200 IU/ml), HEPES (10 mM), and 2-mercaptoethanol(0.1 mM). SKOV-3 cells were labeled with CellTrace Violet (MolecularProbes, Eugene, Oreg.) per the manufacturer's instructions, incubatedwith 5 μg/ml trastuzumab for 30 minutes and washed with the MEM-α basalmedia. NK92 cells and SKOV-3 cells were then resuspended in thesupplemented MEM-α basal media at 1×10⁶ and 2×10⁶/ml, respectively. Fora 1:2 E:T ratio, 100 μl of each cell type was mixed together,centrifuged for one minute at 20×g and incubated at 37° C. for theindicated time points. After each time point, the cells were gentlyvortexed for three seconds and immediately fixed with 4° C. 1%paraformaldehyde in dPBS. Samples were immediately analyzed by flowcytometry. The percentage of conjugated NK cells was calculated bygating on eGFP and CellTrace Violet double-positive events.

To evaluate ADCC, a DELFIA EuTDA-based cytotoxicity assay was usedaccording to the manufacturer's instructions (PerkinElmer, Waltham,Mass.). Briefly, target cells were labeled withBis(acetoxymethyl)-2-2:6,2 terpyridine 6,6 dicarboxylate (BATDA) for 30minutes in their culture medium, washed in culture medium, and pipettedinto a 96-well non-tissue culture-treated U-bottom plates at a densityof 8×10⁴ cells/well. A tumor targeting mAb was added at the indicatedconcentrations of 5 μg/mL and NK cells were added at the indicatedeffector:target (E:T) ratios. The plates were centrifuged at 400×g forone minute and then incubated for two hours in a humidified 5% CO₂atmosphere at 37° C. At the end of the incubation, the plates werecentrifuged at 500×g for five minutes and supernatants were transferredto a 96-well DELFIA Yellow Plate (PerkinElmer, Waltham, Mass.) andcombined with europium. Fluorescence was measured by time-resolvedfluorometry using a BMG Labtech CLARIOstar plate reader (Cary, N.C.).BATDA-labeled target cells alone with or without therapeutic antibodieswere cultured in parallel to assess spontaneous lysis and in thepresence of 2% Triton-X to measure maximum lysis. The level of ADCC foreach sample was calculated using the formula: Percent SpecificRelease=(Experimental release counts−Spontaneous releasecounts)/(Maximal release−Spontaneous release counts)×100%. For eachexperiment, measurements were conducted in triplicate using threereplicate wells.

Statistical Analyses

Statistical analyses were performed by use of GraphPad Prism (GraphPadSoftware, La Jolla, Calif., USA). After assessing for approximate normaldistribution, all variables were summarized as mean±SD. Comparisonbetween two groups was done with Student's t-test, with p<0.05 taken asstatistically significant.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference in their entirety. In theevent that any inconsistency exists between the disclosure of thepresent application and the disclosure(s) of any document incorporatedherein by reference, the disclosure of the present application shallgovern. The foregoing detailed description and examples have been givenfor clarity of understanding only. No unnecessary limitations are to beunderstood therefrom. The invention is not limited to the exact detailsshown and described, for variations obvious to one skilled in the artwill be included within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

SEQUENCE LISTING FREE TEXT rCD64#1 (pCDH-CD64) peptide sequence (SEQ ID NO: 9) MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV LGLQLPTPVW FHVLFYLAVG IMFLVNTVLW VTIRKELKRK KKWDLEISLD SGHEKKVISS LQEDRHLEEE LKCQEQKEEQ LQEGVHRKEP QGAT rCD64#2 (pCDH-mutCD64) peptide sequence (SEQ ID NO: 10) MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV LGLQLPTPVW FHVLFYLAVG IMFLVNTVLW VTIRKELKRK KKWNLEISLD SGHEKKVISS LQEDRHLEEE LKCQEQKEEQ LQEGVHRKEP QGAT rCD64#3 (pCDH-CD64/16) peptide sequence (SEQ ID NO: 11) MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV LGFFPPGYQV SFCLVMVLLF AVDTGLYFSV KTNIRSSTRD WKDHKFKWRK DPQDK rCD64#4 (pCDH-CD64/16-28-BB-Z) peptide sequence (SEQ ID NO: 12) MWFLTTLLLW VPVDGQVDTT KAVISLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV LGFFPPGYQV SFCLVMVLLF AVDTGLYFSV KTNIRSSTRD WKDHKFKWRK DPQDKRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKRGR KKLLYIFKQP FMRPVQTTQE EDGCSCRFPE EEEGGCELRV KFSRSADAPA YQQGQNQLYN ELNLGRREEY DVLDKRRGRD PEMGGKPRRK NPQEGLYNEL QKDKMAEAYS EIGMKGERRR GKGHDGLYQG LSTATKDTYD ALHMQALPPR rCD64#5 (pCDH-CD64/16-28-BB-G) peptide sequence (SEQ ID NO: 13) MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV LGFFPPGYQV SFCLVMVLLF AVDTGLYFSV KTNIRSSTRD WKDHKFKWRK DPQDKRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKRGR KKLLYIFKQP FMRPVQTTQE EDGCSCRFPE EEEGGCELRL KIQVRKAAIT SYEKSDGVYT GLSTRNQETY  ETLKHEKPPQ rCD64#6 (pCDH-CD64/16-28-BB-D10) peptide sequence (SEQ ID NO: 14) MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV LGFFPPGYQV SFCLVMVLLF AVDTGLYFSV KTNIRSSTRD WKDHKFKWRK DPQDKRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKRGR KKLLYIFKQP FMRPVQTTQE EDGCSCRFPE EEEGGCELAR PRRSPAQEDG KVYINMPGRG rCD64#7 (pCDH-CD64/16-28-BB-D12) peptide sequence (SEQ ID NO: 15) MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV LGFFPPGYQV SFCLVMVLLF AVDTGLYFSV KTNIRSSTRD WKDHKFKWRK DPQDKRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKRGR KKLLYIFKQP FMRPVQTTQE EDGCSCRFPE EEEGGCELGR LVPRGRGAAE AATRKQRITE TESPYQELQG QRSDVYSDLN TQRPYYK rCD64#8 (pCDH-CD64-28-28-BB-Z) peptide sequence (SEQ ID NO: 16) MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV LGLQLPTPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKRGR KKLLYIFKQP FMRPVQTTQE EDGCSCRFPE EEEGGCELRV KFSRSADAPA YQQGQNQLYN ELNLGRREEY DVLDKRRGRD PEMGGKPRRK NPQEGLYNEL QKDKMAEAYS EIGMKGERRR GKGHDGLYQG LSTATKDTYD  ALHMQALPPR rCD64#9 (pCDH-CD64-FceR) peptide sequence (SEQ ID NO: 17) MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV LGAPREKYWL QFFIPLLVVI LFAVDTGLFI STQQQVTFLL KIKRTRKGFR LLNPHPKPNP KNN rCD64#10 (pCDH-CD64/16-PKC⁻) peptide sequence (SEQ ID NO: 18) MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV LGFFPPGYQV SFCLVMVLLF AVDTGLYFSV KTNIRGAGRD WKDHKFKWRK DPQDK rCD64#11 (pCDH-CD64-G2D-2B4-Z) peptide sequence (SEQ ID NO: 19) MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV LGSNLFVASW IAVMIIFRIG MAVAIFCCFF FPSGGSGGGS GWRRKRKEKQ SETSPKEFLT IYEDVKDLKT RRNHEQEQTF PGGGSTIYSM IQSQSSAPTS QEPAYTLYSL IQPSRKSGSR KRNHSPSFNS TIYEVIGKSQ PKAQNPARLS RKELENFDVY SGGSGGGSGR VKFSRSADAP AYKQGQNQLY NELNLGRREE YDVLDKRRGR DPEMGGKPRR KNPQEGLYNE LQKDKMAEAY SEIGMKGERR RGKGHDGLYQ GLSTATKDTY  DALHMQALPP R rCD64#12 (pCDH-CD64/16-2B4-Z) peptide sequence (SEQ ID NO: 20) MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV LGFFPPGYQV SFCLVMVLLF AVDTGLYFSV KTNIRSSTRD WKDHKFKWRK DPQDKWRRKR KEKQSETSPK EFLTIYEDVK DLKTRRNHEQ EQTFPGGGST IYSMIQSQSS APTSQEPAYT LYSLIQPSRK SGSRKRNHSP SFNSTIYEVI GKSQPKAQNP ARLSRKELEN FDVYSGGSGG GSGRVKFSRS ADAPAYKQGQ NQLYNELNLG RREEYDVLDK RRGRDPEMGG KPRRKNPQEG LYNELQKDKM AEAYSEIGMK GERRRGKGHD GLYQGLSTAT KDTYDALHMQ ALPPR rCD64#13 (pCDH-CD64/16-2B4-G) peptide sequence (SEQ ID NO: 21) MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV LGFFPPGYQV SFCLVMVLLF AVDTGLYFSV KTNIRSSTRD WKDHKFKWRK DPQDKWRRKR KEKQSETSPK EFLTIYEDVK DLKTRRNHEQ EQTFPGGGST IYSMIQSQSS APTSQEPAYT LYSLIQPSRK SGSRKRNHSP SFNSTIYEVI GKSQPKAQNP ARLSRKELEN FDVYSGGSGG GSGRLKIQVR KAAITSYEKS DGVYTGLSTR NQETYETLKH  EKPPQ rCD64#14 (pCDH-CD64/16-2B4-D10) peptide sequence (SEQ ID NO : 22) MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV LGFFPPGYQV SFCLVMVLLF AVDTGLYFSV KTNIRSSTRD WKDHKFKWRK DPQDKWRRKR KEKQSETSPK EFLTIYEDVK DLKTRRNHEQ EQTFPGGGST IYSMIQSQSS APTSQEPAYT LYSLIQPSRK SGSRKRNHSP SFNSTIYEVI GKSQPKAQNP ARLSRKELEN FDVYSGGSGG GSGARPRRSP AQEDGKVYIN MPGRG rCD64#15 (pCDH-CD64/16-2B4-D12) peptide sequence (SEQ ID NO: 23) MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV LGFFPPGYQV SFCLVMVLLF AVDTGLYFSV KTNIRSSTRD WKDHKFKWRK DPQDKWRRKR KEKQSETSPK EFLTIYEDVK DLKTRRNHEQ EQTFPGGGST IYSMIQSQSS APTSQEPAYT LYSLIQPSRK SGSRKRNHSP SFNSTIYEVI GKSQPKAQNP ARLSRKELEN FDVYSGGSGG GSGGRLVPRG RGAAEAATRK QRITETESPY QELQGQRSDV  YSDLNTQRPY YK rCD64#16 (pCDH-CD64-64-2B4-Z) peptide sequence (SEQ ID NO: 24) MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV LGLQLPTPVW FHVLFYLAVG IMFLVNTVLW VTIRKELKRK KKWDLEISLD SGHEKKVISS LQEDRHLEEE LKCQEQKEEQ LQEGVHRKEP QGATGWRRKR KEKQSETSPK EFLTIYEDVK DLKTRRNHEQ EQTFPGGGST IYSMIQSQSS APTSQEPAYT LYSLIQPSRK SGSRKRNHSP SFNSTIYEVI GKSQPKAQNP ARLSRKELEN FDVYSGGSGG GSGRVKFSRS ADAPAYKQGQ NQLYNELNLG RREEYDVLDK RRGRDPEMGG KPRRKNPQEG LYNELQKDKM AEAYSEIGMK GERRRGKGHD GLYQGLSTAT KDTYDALHMQ ALPPR Canine CD16#17 (pCDH-caCD16) peptide sequence (SEQ ID NO: 25) MWQLVSSTAL LLLVSAGTQA DVPKAVVVLE PKWNRVLTMD SVTLKCQGDH LLRDNYTWLH NGRPISNQIS TYIIKNASIK NSGEYRCQTD QSKLSDPVQL EVHTGWLLLQ VPRLVFQEGE LIQLKCHSWK NTPVRNVQYF QNGRGKKFFY NNSEYHIPAA TSEHNGSYFC RGIIGKKNES SEAVNIIIQG SSLPSTSLLL SHWPQIPFSL VMALLFAVDT GLYFAVQRDL RSSMGNLKNS KVIWSQGS Canine CD64#18 (pCDH-caCD64) peptide sequence (SEQ ID NO: 26) MWLLTVLLLW VPAGAQTDPV KAVITLQPPW VSVFQEESVT LWCEGPHLPG DSSTQWFLNG TATQTLTPRY RIAAASVNDN GEYRCQTGLS VLSDPIQLGI HRDWLILQVS GRVFTEGEPL TLRCHGWNNK LVYNVLFYQN GTVLKFSPQN SEFTILKTTL HHNGIYHCSA MGKHRYESAG VSITIKELFP APVLKASLSS PILEGHVVNL SCETKLLLQR PGLQLYFSFY MGSKTLLSRN TSSEYQILTA KKEDSGLYWC EATTEDGNVV KRSPELELQV VGPQTLTPVW FHVLFYVAMG MIFLVDTIFC MIIHKELQRK KKWNLEISLY SGLEKRVDSY LQKERDLEEP KYQELEQLQE KTPQKPPEGE QQ Canine CD64sp#19 (pCDH-caCD64sp) peptide sequence (SEQ ID NO: 27) MWLLTVLLLW VPAGAQTDWL ILQVSGRVFT EGEPLTLRCH GWNNKLVYNV LFYQNGTVLK FSPQNSEFTI LKTTLHHNGI YHCSAMGKHR YESAGVSITI KELFPAPVLK ASLSSPILEG HVVNLSCETK LLLQRPGLQL YFSFYMGSKT LLSRNTSSEY QILTAKKEDS GLYWCEATTE DGNVVKRSPE LELQVVGPQT LTPVWFHVLF YVAMGMIFLV DTIFCMIIHK ELQRKKKWNL EISLYSGLEK RVDSYLQKER DLEEPKYQEL EQLQEKTPQK PPEGEQQ 

What is claimed is:
 1. A chimeric IgG Fc receptor comprising: anextracellular domain comprising a sufficient portion of CD64 to bind toan IgG Fc region; a transmembrane domain; and an intracellular domaincomprising a sufficient portion of an Fc receptor immunoreceptortyrosine-based activation motif (ITAM) to initiate cell signaling whenan IgG Fc region binds to the extracellular domain.
 2. The chimeric IgGFc receptor of claim 1, wherein the intracellular domain comprises atleast a portion of the intracellular region of CD16A.
 3. The chimericIgG Fc receptor of claim 1, wherein the intracellular domain comprisesat least a portion of the intracellular region of CD27, CD28, CD134(OX40), CD137 (4-1BB), FcεR1, NKG2D, CD244 (2B4), FcRγ, DAP10, DAP12, orCD3ζ.
 4. The chimeric IgG Fc receptor of any preceding claim, whereinthe extracellular domain comprises the CD16A cleavage site.
 5. Thechimeric IgG Fc receptor of any preceding claim, wherein theintracellular domain comprises a signaling domain.
 6. A polynucleotideencoding the chimeric receptor of any preceding claim.
 7. A recombinantcell comprising the polynucleotide of claim
 6. 8. A recombinant cellexpressing the IgG Fc chimeric receptor of any one of claims 1-5.
 9. Therecombinant cell of claim 8, wherein the recombinant cell is a naturalkiller (NK) cell.
 10. A recombinant natural killer (NK) cell comprisinga polynucleotide that encodes CD64.
 11. A recombinant cell comprising anatural killer (NK) cell genetically modified to express CD64.
 12. Amethod of killing a tumor cell, the method comprising: contacting thetumor cell with an antibody that specifically binds to the tumor cell;and contacting the tumor cell with the recombinant cell of any one ofclaims 7-11 under conditions effective for the recombinant cell to killthe tumor cell.
 13. A method of treating a subject having a tumor, themethod comprising: administering to the subject an antibody thatspecifically binds to cells of the tumor; and administering to thesubject a composition comprising the recombinant cell of any one ofclaims 7-11 under conditions effective for the recombinant cell to killcells of the tumor.
 14. A composition comprising: the recombinant cellof any one of claims 7-11; and an antibody bound to the chimericreceptor.
 15. A method of treating a subject having a tumor, the methodcomprising: administering to the subject the composition of claim 14wherein the antibody specifically binds to cells of the tumor.